Anti-methicillin-resistant Staphylococcus aureus and antibiofilm activity of new peptides produced by a Brevibacillus strain.

Background: Methicillin-resistant Staphylococcus aureus (MRSA) is listed as a highly prioritized pathogen by the World Health Organization (WHO) to search for effective antimicrobial agents. Previously, we isolated a soil Brevibacillus sp. strain SPR19 from a botanical garden, which showed anti-MRSA activity. However, the active substances were still unknown. Methods: The cell-free supernatant of this bacterium was subjected to salt precipitation, cation exchange, and reversed-phase chromatography. The antimicrobial activity of pure substances was determined by broth microdilution assay. The peptide sequences and secondary structures were characterized by tandem mass spectroscopy and circular dichroism (CD), respectively. The most active anti-MRSA peptide underwent a stability study, and its mechanism was determined through scanning electron microscopy, cell permeability assay, time-killing kinetics, and biofilm inhibition and eradication. Hemolysis was used to evaluate the peptide toxicity. Results: The pure substances (BrSPR19-P1 to BrSPR19-P5) were identified as new peptides. Their minimum inhibition concentration (MIC) and minimum bactericidal concentration (MBC) against S. aureus and MRSA isolates ranged from 2.00 to 32.00 and 2.00 to 64.00 µg/mL, respectively. The sequence analysis of anti-MRSA peptides revealed a length ranging from 12 to 16 residues accompanied by an amphipathic structure. The physicochemical properties of peptides were predicted such as pI (4.25 to 10.18), net charge at pH 7.4 (-3 to +4), and hydrophobicity (0.12 to 0.96). The CD spectra revealed that all peptides in the water mainly contained random coil structures. The increased proportion of α-helix structure was observed in P2-P5 when incubated with SDS. P2 (NH2-MFLVVKVLKYVV-COOH) showed the highest antimicrobial activity and high stability under stressed conditions such as temperatures up to 100 °C, solution of pH 3 to 10, and proteolytic enzymes. P2 disrupted the cell membrane and caused bacteriolysis, in which its action was dependent on the incubation time and peptide concentration. Antibiofilm activity of P2 was determined by which the half-maximal inhibition of biofilm formation was observed at 2.92 and 4.84 µg/mL for S. aureus TISTR 517 and MRSA isolate 2468, respectively. Biofilm eradication of tested pathogens was found at the P2 concentration of 128 µg/mL. Furthermore, P2 hemolytic activity was less than 10% at concentrations up to 64 µg/mL, which reflected the hemolysis index thresholds of 32. Conclusion: Five novel anti-MRSA peptides were identified from SPR19. P2 was the most active peptide and was demonstrated to cause membrane disruption and cell lysis. The P2 activity was dependent on the peptide concentration and exposure time. This peptide had antibiofilm activity against tested pathogens and was compatible with human erythrocytes, supporting its potential use as an anti-MRSA agent in this post-antibiotic era.

Purification and Characterization of Novel Anti-MRSA Peptides Produced by Brevibacillus sp. SPR-20.

Methicillin-resistant Staphylococcus aureus (MRSA) is listed as a high-priority pathogen because its infection is associated with a high mortality rate. It is urgent to search for new agents to treat such an infection. Our previous study isolated a soil bacterium (Brevibacillus sp. SPR-20), showing the highest antimicrobial activity against S. aureus TISTR 517 and MRSA strains. The present study aimed to purify and characterize anti-MRSA substances produced by SPR-20. The result showed that five active substances (P1-P5) were found, and they were identified by LC-MS/MS that provided the peptide sequences of 14-15 residues. Circular dichroism showed that all peptides contained ß-strand and disordered conformations as the major secondary structures. Only P1-P4 adopted more α-helix conformations when incubated with 50 mM SDS. These anti-MRSA peptides could inhibit S. aureus and MRSA in concentrations of 2-32 µg/mL. P1 (NH2-VVVNVLVKVLPPPVV-COOH) had the highest activity and was identified as a novel antimicrobial peptide (AMP). The stability study revealed that P1 was stable in response to temperature, proteolytic enzymes, surfactant, and pH. The electron micrograph showed that P1 induced bacterial membrane damage when treated at 1× MIC in the first hour of incubation. The killing kinetics of P1 was dependent on concentration and time. Mechanisms of P1 on tested pathogens involved membrane permeability, leakage of genetic material, and cell lysis. The P1 peptide at a concentration up to 32 µg/mL showed hemolysis of less than 10%, supporting its safety for human erythrocytes. This study provides promising anti-MRSA peptides that might be developed for effective antibiotics in the post-antibiotic era.